QUICK QUALITY TESTS FOR PROTEIN MEALS

Dr. Jowaman Khajarern and Dr. Sarote Khajarern
Department of Animal Science, Faculty of Agriculture
Khon Kaen University, Thailand


INTRODUCTION

Since the early 1970s, the feed industry in Southeast Asia has made tremendous expansion. High quality materials especially protein meals (fish meal and soybean meal) are often in short supply and also exhibit a variation in the quality aspects of nutrient availability. Since protein sources especially fish meal and soybean meal generally have high unit cost, the company must establish written ingredient quality standards for purchasing, but the methods for examining the physical qualities, especially for foreign materials and evidence of mold, must be fast, accurate and practical by the operators at the receiving plant. The operators must be trained to recognize and understand the quality of raw material to perform their visual and other quick physical and chemical examinations, and in the proper methods of sampling. The objective of this paper is to highlight the most important method of quick tests for protein meals used for the purchasing of the raw materials.

Sources of Protein Meals for Non-ruminants and Aquaculture

Sources			Plant Protein			Animal Protein
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Conventional Major	Soybean meal			Fish meal
			Fullfat soybean			Meat meal
			Sunflower meal			Meat and bone meal
			Sesame meal			Milk products
			Peanut meal			Poultry byproducts
			Canola and Rapeseed meal
			Corn gluten meal
			Palm kernel meal
			Copra meal	
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Minor			Soya protein isolate		Feather meal
			Wheat gluten meal		Blood meal
			Mung bean			Marine soluble products
			Potato protein			Shrimp products
			Cotton seed meal		Squid products
			Kapok seed meal			Yeast

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Non-conventional	Rubber seed meal		Leather meal
			Lupins				Silk worm pupa meal
			Safflower meal			Crab products
			Linseed meal			Lard pulp
			Field peas			Brewer's byproducts
			Mustard meal			Distiller's byproducts
			Cocoa seed meal	

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Quality Control in Different Protein Meals
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Ingredient Quality	Physical characteristics (analyst's skills): color, texture, odor and 
(Qualitative)		taste, particle size (screen analysis), shape, adulteration, damage      
         		and deterioration, bulk density, spot and quick chemical tests.     
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Ingredient Quality	Proximate analysis: moisture, CP, CF, EE, NFE, ash, silica or
(Quantitative)      		sand, salts etc.
             		Protein quality: protein solubility or dispersibility, bitrogen 
                    		solubility, mailard reaction product, biogenic amines, trypsin 
               			inhibitor activity, urease activity, dye binding, pepsin 
                         	digestibility, urea and nonprotein nitrogen.
          		Amino acid: composition, digestibility, availability
          		Anti-nutritional factors:
                        -     Extrinsic (contaminants): mycotoxins, insects, weeds, 
                              insecticide, herbicides, fungicides.
                        -     Intrinsic: allergins, lectins, phytoestrogens, 
                              glucosinolate, saponins, tannins, ricin, sinapine, 
                              gossypol, lipoxygenase.
			Decomposition and rancidity test: acid value, peroxide value, etc.
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Quick Test for Fish Meal Quality Control

Basically, fish meal is produced from two types of raw materials, fish wastes from human food industry and whole fish. Differences in composition of fish meal may be attributed to many causes: variation in raw materials, differences in processes, contamination of raw materials with some waste products or with sand, impurities, excessive salts and/or fat content or excessive moisture, adulteration with other sources of meal, meat and bone meal, plant proteins, etc. Fish meal is also prone to contamination with biogenic amine and gizzarosine found during processing and storage that have been allowed to spoil or putrefy and have dramatic impact on the quality and nutritive composition of fish meal. The main aspects of quality control for consideration are:

  • Raw material type: whole fish or trimmings

  • Processing temperature: low temperature (LT) high temperature (HT) on digestibility of protein and over-heated by using racemization of aspartic acid (Luzanna et al., 1996) to indicate digestibility of protein and a simple dilute pepsin test (strength 0.0002 %) at 450C will identify overheated fish meal.

  • Freshness of raw material: Fish spoils protein breakdown to amines (histamine, cadaverine putrescine and tyramine). The sum of the four should be less than 2,000 ppm or predominant in anchovy, mackerel, sardine, etc. or cadaverine predominant in capelin, sandeel, sprat, etc).

  • Lipid quality: Oxidation of fat to free fatty acids given a crude measure of fish freshness. Oxidation of fat can reduce growth of animal and aquaculture. Ethoxyquin has been found to be the most effective antioxidant. Free fatty acid (FFA) peroxide value (PV) and quick test for rancidity should be assessed for quality control.

  • Microbial standards: Fish meal should be free of salmonella and mycotoxin. Because carbohydrate or starch is absent, mold growth generally does not occur if fish meal is properly stored in dry conditions and stabilized with antioxidants.

Quick Test for Quality Grade of Fish Meal

  1. Place 10g of fish meal in a 100 ml tall form beaker and pour 80 ml carbon tetrachloride into the beaker, stir and allow to settle (5 min.).
  2. Scrape off the floating organic fraction with stainless steel spoon into filter paper. Clean off the side of the beaker and spoon and then pour off the liquid to separate floating and the submerged fraction into the filter paper.
  3. Place the two fractions into a 1100C oven for 10 min. then allow to cool and weigh each fraction.
  4. The approximate percentage of organic (fish flesh) and inorganic (fish bone) may be calculated.
	Shrimp and Salmon Grade     	     Inorganic Fraction
     	Fish meal Grade A (CP>70%)               <12%
     	Fish meal Grade B (CP>65%)               <16%
    	Fish meal Grade C (CP>62%)               <20%

	Animal Grade                         Inorganic Fraction
     	Fish meal Grade A (CP>58%)               <30%
    	Fish meal Grade B (CP>35-58%)            <35%
   	Fish meal Grade C (CP>50-55%)            <40%
   	Fish and bone meal (CP 35-50%)		 >40%

Quick Test for Quality Grade of Fish Meal

Principle:

Most adulterants such as (ammonium sulfate, ammonium phosphate, ammonium nitrate, etc) are non-protein nitrogen and are soluble in water. When they react with mercuric potassium iodide alkaline solution mixture, a heavy orange precipitate color occurs.

Reagent:

1.25g mercuric chloride is made into a thin paste with a little water, 3.5 g potassium iodide is added and solution takes place. 12 g sodium hydroxide is dissolved in 50 ml. Water is added and the whole made up to 100 ml. The rather turbid solution is allowed to stand for several days, decanted and stored in a brown bottle.

Procedure:

  1. Put 2-3 g of test sample in a 100 ml beaker and add 10-15 ml distilled water, then stir and let stand for 2-3 min.
  2. Put 3-5 drops of test sample into white porcelain spot plates and add 2-3 drops of mercuric-potassium iodide alkaline solution mixture.
  3. If non-protein nitrogen is present, a heavy orange precipitation color appears. The intensity of orange precipitation depends on the amount of non-protein nitrogen present.

Quick Test of Hydrolyzed Feather Meal from Fish Meal

Principle:

Hydrolyzed feather meal contains a high percentage of cystine (6-7%). When the sample is digested with sodium hydroxide, the cystine and cysteine are liberated and reaction with lead acetate gives the dark brown black color on the surface of the particle.

Reagents:

Solution A: Sodium hydroxide 10%

Solution B: Dissolve 50 g of lead acetate in 800 ml water, then add 20 ml glacial acetic acid, stir and pour 20 ml glucerol, shake and dilute with water to 1,000 ml.

Procedure:

  1. Place one teaspoon of well mixed standard hydrolyzed feather meal and test sample of fish meal into two sets of petri dishes.
  2. Add 10-15 ml of solution A into all the two sets of each test sample. Swirl gently to spread samples evenly in each dish and let them stand for 10 min.
  3. Add 10-15 ml of solution B into each first set of petri dishes and into the second set add 10-15 ml distilled water. Mix gently by turning around each petri dish and let them stand again for 10 min.
  4. During standing, a visible browning reaction color develops until black colored particles appear in the first set of petri dish for the standard hydrolyzed feather meal. When compared to the second set (without adding solution B), no visible brown color develops after 10 mins.
  5. Compare the test fish meal sample with the standard hydrolyzed feather meal sample and also the visible browning color between the first and second set of each test sample. If the color of these two sets differs, the fish meal is adulterated with hydrolyzed feather meal.

Quick Test of Hydrolyzed Leather Meal from Fish Meal

Leather meal is waste by-products from hide trimmings which contain chromium salts. When a sample is ashed, the visible dark green chromium salts develops. Diphenyl carbazide is an excellent reagent to react with chromium in dilute sulfuric acid (0.1 MH2SO4) which produces a violet color and will fade within 10-15 mins.

Reagents:

Solution A: (0.1 MH2SO4
Solution B: Dissolve 1-2 g diphenyl carbazide in 100 ml distilled water.

Procedure:

  1. Ash fish meal test sample at 6000C for 2 hrs and examine for the presence of dark green chromium salts. Dark green particle of ash is a conducive evidence of the adulteration of leather meal in the sample.
  2. To confirm the presence of chromium, place a small amount of ash into a white spot plate. Add 2-3 drops of solution A and then add 2-3 drops of solution B.
  3. A red-violet color indicates the presence of chromium.

Decomposition Test for Animal and Marine Products

Principle:

As animal and marine products spoil, protein breaks down to amines. The residue of these biogenic amine can indicate the freshness or decomposition of the sample. If the sample is badly decomposed, the test sample will darken quickly with saturated lead acetate paper and it is not suitable for feeding.

Reagents:

  1. Dilute sulfuric acid: 5 ml of conc. H2SO4 to 45 ml H2O
  2. Saturated lead acetate solution.

Procedure:

  1. Put 5 g of test sample into 250 ml erlenmeyer flask.
  2. Prepare a cork, which fits tightly, with a 2" x ¼" strip of white filter paper pinned to the bottom, moistened with saturated lead acetate.
  3. Add 50 ml dilute sulfuric acid into the sample then immediately insert the cork and let it stand in a warm room for 16 hrs.
  4. If the sample is badly decomposed, the test paper will darken quickly.

Quick Test for Identification of Plant Protein and Animal Protein

Principle:

Carbohydrates from plants contain starch and cellulose. When it reacts with iodine and chlor-zinc iodine solution, the starchy tissue releases a blue color and the plant fiber or cellulose develops a purple-brown color when examined under a microscope.

Reagents:

  1. Iodine solution: 0.5 g I and 1.5 g KI dissolved in 25 ml distilled water.
  2. Chlor-zinc-iodine solution: Dissolve 100 g ZnCl2 in 60 ml water, then add 20 g KI and 0.5 g I.

Procedure:

  1. Mix 1-2 g test sample with 100 ml boiling water or boil the mixture for 2-3 min. Place a few ml of the cooled mixture on a spot plate or test tube and add 5-6 drops of iodine solution. If starch is present, the mixture turns blue.
  2. Spread 1-2 g test sample into a petri dish. Add 5-6 drops of chlor-zinc iodine solution and let stand for 10 min. A purple brown color indicates the presence of plant fiber, whereas yellow indicates animal fiber (protein) using a microscopic examination.

Quality Considerations for Soybean Meal

Soybean meal is a major plant protein source for poutlry, livestock and aquaculture. Soybean meal used in Asian countries exists in several forms, with solvent extracted material containing hulls being the most common. Many locations use imported soybean meal from USA, Brazil, Argentina and India. Full fat soybean is also available, produced by extrusion or dry roasting in small scale plants. The following are specifications for soybean meal and dehulled soybean meal (Hi-Pro).

  • Bulk Density Range: 57-64 g/100 cc for both soybean meal and soybean (Hi-Pro).
  • Screen Analysis: 95-100% through US #10 screen; 40-60% through US #20, 6% maximum through US #80 for both soybean and soybean (Hi-Pro).
  • Desired Physical Properties for both soybean and soybean (Hi-Pro).
    • Color: Light tan to light brown
    • Odor: Fresh, not musty, not sour or burned
    • Texture: Homogeneous, free flowing, no lumps or cakes, without coarse particles or dusty
    • Taste: Bland and free of any beany or burned taste
  • Urease actvity: 0.05-0.20 pH unit change for both soybean meal and soybean (Hi-Pro).
  • Moisture (maximum): 12% for both soybean meal and soybean (Hi-Pro).
  • Protein solubility 0.2% KOH: 73-85% for both soybean meal and soybean (Hi-Pro).
  • Protein Dispersibility Index: 15-30% for both soybean meal and soybean (Hi-Pro).
  • Nitrogen Solubility Index: 15-30% for both soybean meal and soybean (Hi-Pro).
  • Contaminants: Particularly check for urea, non-protein nitrogen or ammonia for both soybean meal and soybean (Hi-Pro).
  • Protein (minimum): 44.0% for soybean meal; 47.5-49% for soybean (Hi-Pro).
  • Fat (minimum): 0.5% for both soybean meal and soybean (Hi-Pro).
  • Fiber (maximum): 7.0% for soybean meal; 3.3-3.5% for soybean (Hi-Pro).
  • Ash (maximum): 6% for both soybean meal and soybean (Hi-Pro).
  • Silica (maximum): 2% for both soybean meal and soybean (Hi-Pro).
  • ME ((kcal/kg): Approximately 2,375 for soybean meal; 2,525 for soybean (Hi-Pro).

Rancidity test for Animal Products and Oilseed Meal

Method I

Reagents:

  1. Acetic chloroform mixture (6:4): Glacial acetic acid 60 ml and 40 ml chloroform.
  2. Saturated potassium iodide.
  3. Starch indicator.

Procedure:

  1. Put 5g test sample into 250 ml Erlenmeyer flask.
  2. Add 50 ml of acetic chloroform mixture and 1 ml saturated potassium iodide and shake.
  3. Add 50 ml distilled water and starch indicator, then shake again.

The development of blue color indicates rancidity.

Method II

Reagents:

  1. 0.1% Phloroglucinal solution in ether.
  2. Kerosene.

Procedure:

  1. Shake 10 ml of oil or melted fat sample with 10 ml of 0.1% phloroglucinal solution and 10 ml of conc. Hydrochloric acid for 20 sec. A pink color indicates incipient rancidity.
  2. If oil is diluted 1 in 20 kerosene and the test is still positive, the rancidity will probably be evident to the taste and smell.

Quick Test for Urea (Qualitative Test)

Reagents:

  1. Urease solution: 0.2 g urease stir into 10 ml H2O.
  2. Bromothymol blue solution: Rub 0.15 g Bromothymol blue in mortar with 2.4 ml 0.1 N NaOH, wash mortar and pestle with H2O and dil. To 50 ml H2O.
  3. Test paper A: Mix 10 ml urease solution 1 and 10 ml indicator soln. 2. Pour mixture into watch glass, dip pieces of filter paper (Whatman No. 5) in soln. And hang paper to dry. Store dry paper (Orange color) in well-stoppered dark glass bottle in a cool place.
  4. Test paper B: Dilute indicator soln. 2 with equal portion with H2O. Dip pieces of filter paper (same kind used for test paper A) in indicator soln. And hang to dry as in 3.

Procedure:

  1. Stir 2-3 g of test sample in 50 ml H2O and let it stand for 2-3 min.
  2. Placing 2-3 drops of test sample on dry test paper A, the appearance of blue or green spot after a few minutes of incubation at room temperature indicates urea.
  3. For detection of urea in a very small, dry particle, dip both test paper A and B H2O and then shake the papers to remove excess H2O by using clean tweezers. Place the papers on a clean flat piece of glass. Place the sample on the papers and cover with another clean flat piece of glass by pressing down gently. Blue spots on the test paper A indicate urea (30-60 sec.). Spots continue to develop and enlarge for 10-20 min. and then fade gradually. Time varies inversely with urea concentration. If blue spots develop on both paper A and B, this indicates alkaline particles.

Microscopic Identification of Protein Meals

Feed microscopy experts must be fast and accurate to obtain quality assurance. Adulterants and contaminants in both ingredients and finished feeds are best detected microscopically. Special training on description and characteristics of feed ingredients are the keys for rapid microscopic identification.

Preparation of sample

If the feed is mashed, use about a teaspoonful of feed for sieving. Rub the feed in a mortar with a pestle if the feed is pelleted or crumbled. When the sample is reduced to a mash-like consistency, it is divided in three sets of sieves (20, 40, 60 mesh), and thus end up with four fractions of the feeds. Each fraction is put in a glass petri dish cover. The sample is now ready for microscopic examination. Using a stereomicroscope, scan each particle in the first and second dishes. The feed in the third and fourth dishes is used for high power compound microscope and also for quick chemical spot test. For a clearer observation of plant histology and microscopic appearance, the feed is heated with 8% KOH steam bath for 30-45 min. If this treatment is not satisfactory, treat the fresh portion for a short time by gently heating with acidified chloral hydrate glycerol solution.

Precise characteristics on Microscopic Identification of Protein Meals
(Contact us for a copy of the annex picture).

  • Crab Products: Orange pigmentation, segmented antennae with calcareous shell and will effervesce in diluted HCI, honey combed round cells on the outer layer of shell.

  • Fish Products: Curved scales with concentric rays, bone sexhibit lacunae with well defined canaliculi, milky glass beads with broken surface eye lenses.

  • Shrimp Meal: Segmented leg and antennae, thin shell with mica-like and in some areas, may appear cross-hatch type of marking, feathery delicate gill tissue, amber colored cells of compound eyes.

  • Squid Products: Mottled body fragment with black pigment spots, tentacles or sucker pieces present, no lacunae or surface lines on internal shell fragments.

  • Blood Meal: Spherical particle, smooth surface with glass when rubbed, dark red to almost black in color.

  • Meat Meal and Meat and Bone Meal: Strong greasy odor, consist of hoof, horn, hair, fluff and vegetable fiber, cylindrical rods smooth muscle with alternative dark and light striated muscle.

  • Soybean Meal: Yellow to brown oval hilium with a clear slit, pox-marked outer surface hull, hourglass and palisade cells from the hull are the major cellular keys for soybeans and also elongated cells below the peripheral cells of the cotyledon.

  • Peanut Meal: Thin skin with copper to red color, higly pitted cell of pod fragment and the lack of palisade cells in the testa, elongated pite in hypodermal stone cells and unique cross fiber cell of pod.

  • Sunflower Meal: Striped or all black varieties for the hull, leathery hull with a paper-like lining. Twin hairs, united almost to their tips on the outer surface of the cypsela, unbroken pericarp fragment may appear as broken pieces in the medium, the outer epiderm of transversely elongated cells with zigziag walls.

  • Rapeseed Meal: Many species of rape are lumped together and are difficult to identify separately by structured features. For all practical purposes, the examination of the seed coat or testa for degree of reticulation is important. The inner surface seed coat has a delicate semi-transparent, white sheet adhering to the surface.

  • Sesame Meal: Seed coat or outer epidermal cells contain calcium oxalate crystals with black brown or yellow brown colored and granular surface.

  • Cottonseed Meal: Long, flat and twisted fibers adhering to the hulls, kernel fragments are yellow to brown containing many round, red, brown gossypol glands. The hull edge has a light brown layer with stairstep facets. The epidermal cells are heavy walled with dark pigmented interiors. Palisade cells can also be used for identification.

  • Copra Meal: Irregularly shaped flaky fragment with large, colorless, straight, thin walled cells of endosperm containing oil globules.

REFERENCES:

  1. Association of Official Analytical Chemists, 1984. Official Methods of Analysis. 12th Edition, AOAC, Box 540, Benjamin Franklin Station, Washington DC 20044.

  2. Khajarern, J., D. Sinchermsiri, and A. Hanbunchong, 1987. Manual Feed Microcopy and Quality Control. Dhornhvaj Co., Ltd. Bangkok, Thailand.

  3. Luzzana, U., T. Mentasti, V. Moretli, A. Albertini, and E. Valfre, 1996. Aspartic acid raccimization in fish meal as induced by thermal treatment. Aquac. Nutr. 2: 95-99.

  4. Manual of Microscopic Analysis of Feeding Stuffs, 1966. The American Association of Feed Microscopists.

  5. Manual of of Microscopial Analysis of Feedstuff, 1992. 3rd Edition, The American Association of Feed Microscopists.

  6. Peason, D., 1970. The Chemical Analysis of Foods, 6th Edition. J & A Churchill, 164 Gloucester Place, London.

AMERICAN SOYBEAN ASSOCIATION
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Singapore 238881.
TEL: (65) 6737-6233, FAX: (65) 6737-5849.

URL: http://www.asasea.com

MITA (P) NO. 096/11/97 (Vol. FT45-1998)
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